Animal and Plant Transformation: The Application of Transgenic Organisms in Agriculture
Matthew B. Wheeler, Stephen K. Farrand, and Jack M. Widholm
A transgenic organism carries in all its cells a foreign gene that was inserted by laboratory techniques. Each transgenic organism is produced by introducing cloned genes, composed of deoxyribonucleic acid (DNA) from microbes, animals, or plants, into plant and animal cells. Transgenic technology affords methods that allow the transfer of genes between different species.
Through transgenic animal transformation, new genetic information is introduced into an animal in one generation without compromising or limiting the overall pool of genetic information. Transgenic animals are produced by inserting genes into embryos prior to birth. Each transferred gene is assimilated by the genetic material or chromosomes of the embryo and subsequently can be expressed in all tissues of the resulting animal. The objective is to produce animals which possess the transferred gene in their germ cells (sperm or ova). Such animals are able to act as "founder" stock to produce many offspring that carry a desirable gene or genes.
Transgenic animals have been produced by three methods: microinjection of cloned gene(s) into the pronucleus of a fertilized ovum, injection of embryonic stem cells into embryos, and exposure to retroviruses. The third method is not discussed in this article.
The first method is the one that is most widely and successfully used for producing transgenic mice. After microinjection, the recently fertilized single cell embryos are removed from the animal. Micromanipulators on a specially equipped microscope are used to grasp each embryo. A glass pipette drawn or pulled to a fine point immobilizes the embryo on one side, as shown in the photos to the right. On the opposite side, the foreign DNA is injected into the embryo's pronucleus--either of two nuclei (male or female) containing half the chromosomes of a fertilized ovum--with a second finely drawn injection needle. After the injection, the embryos are transferred back into the hormonally prepared or pseudopregnant recipient females or foster mothers. The recipients follow normal pregnancy and deliver full-term young. This method is presently the most efficient for generating transgenic animal lines: about 1 to 4 percent of the injected embryos result in a transgenic offspring.
The second method involves microinjection of embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos (about 7 days postfertilization) into embryos to produce "hybrid" embryos of two or more distinct cell types. The ES cells are able to produce all tissues of an individual. Once isolated, ES cells may be grown in the lab for many generations to produce an unlimited number of identical cells capable of developing into fully formed adults. These cells may then be altered genetically before being used to produce embryos. When these transformed cells participate in the formation of sperm and eggs, the offspring that are produced will be transgenic. Results have shown this method to be promising for producing transgenic mice. Studies are presently under way at the University of Illinois Department of Animal Sciences to develop ES cell lines for livestock species such as swine, cattle, and sheep.
To produce transgenic mice, Matthew B. Wheeler microinjects DNA into the pronucleus of one-cell embryos.
Injection of cloned DNA into embryos. One-cell embryo is positioned for micro-injection into the pronucleus (left). The plasma membrane has been pierced, and the tip of the needle remains inside the pronucleus, while DNA is expelled from the needle, causing the pronucleus to swell visibly.
These methods, which enable the insertion of foreign genes into embryos, have provided the tools for producing new strains or breeds of animals that carry new, beneficial genetic information. These technologies do not produce new species but work within the established genetic framework of existing species to improve them. Some new strains developed include leaner, more feed-efficient, faster-growing swine containing additional copies of the growth hormone gene, and mice containing the regulatory elements of the human immunodeficiency virus (HIV) genome. The latter are used as a noninfectious animal model for the study of AIDS.
The scope of the information acquired from transgenic animal technology is pertinent to virtually all areas of modern agriculture and biomedical science--cancer research; immunology; developmental biology; gene expression and regulation; and models for human genetic diseases such as muscular dystrophy, Lou Gehring's disease, and sickle cell anemia. Potential applications for transgenic animals include manipulation of milk composition, growth, disease resistance, reproductive performance, and production of pharmaceutical proteins by livestock.
There has been much excitement in the last few years about our ability to genetically engineer plants using the new techniques of gene isolation and insertion. Paired with standard methodologies of plant tissue culture and plant regeneration, these new techniques allow us to construct transgenic plants that contain and express a single, well-defined gene from any source - microbe, animal, or other plant species. The transgenic plants, usually normal in appearance and character, differ from the parent only with respect to the function and influence of the inserted gene.
This directed genetic engineering of plants requires that genes of interest are available, that the gene be introduced into plant cells capable of regenerating into intact plants, and that the gene carries with it a selectable marker so that the transformed plant cells can be isolated from a large population of untransformed, normal cells. Finally, the transformed plant cell must retain its capacity to regenerate. Certain species such as tobacco and petunia regenerate plants quite easily, making transgenic plants readily obtainable. Although corn, soybean, and wheat--the primary agricultural crops of Illinois and the Midwest--are more recalcitrant to these manipulations, progress is being made toward routine transformation and regeneration of transgenic progeny of these species.
Several techniques can introduce genes into plant cells. Perhaps the most successful method involves the pathogenic bacterium Agrobacterium tumefaciens, which has the innate ability to transfer DNA to plant cells. In nature, this transfer results in formation of plant tumors (crown galls) at the infection site. Molecular biologists, however, have disarmed this bacterium and constructed domesticated strains that no longer cause tumors but transfer any DNA of interest to plant cells. The major disadvantage of the highly efficient Agrobacterium system is that it does not work with all plant species, most notably the cereals.
Other techniques use physical or chemical agents to transfer DNA into plant cells. Protoplasts, plant cells that have been stripped of their protective cell walls, will take up pure DNA when treated with certain membrane-active agents or with electroporation, a rapid pulse of high-voltage direct current. Once inside the cell, the DNA is integrated and the foreign gene will express. These two techniques largely depend upon the development of protoplast systems that retain the capacity to regenerate intact plants. Transgenic corn, rice, and soybean have been produced with these techniques, especially electroporation. Success rates, however, are low, and the techniques not very reproducible.
DNA can also be microinjected into target plant cells using very thin glass needles in a method similar to that used with animals. Microinjection, however, has produced only a few transgenic plants. The technique is laborious, technically difficult, and limited to the number of cells actually injected.
Biolistics, a new method, involves accelerating very small particles of tungsten or gold coated with DNA into cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the particles pass through the cell, the DNA dissolves and becomes free to integrate into the plant-cell genome. This improbable technique actually works quite well and has become, along with electroporation, one of the methodologies of choice. Biolistics has the advantage of being applicable to whole cells in suspension or to intact or sliced plant tissues. For example, plant meristems or tissues capable of regeneration can be targeted directly. Unlike transformation or electroporation, the technique does not require protoplasts or even single-cell isolations. Using biolistics, transgenic corn and soybean plants have been produced that contain heritable copies of the inserted gene.
Only a few genes of agronomic importance have been inserted into plants: genes conferring resistance to certain insects and viruses and also those conferring tolerance to broad-spectrum herbicides. The latter result in increased herbicide specificity, allowing the farmer to use more effective, environmentally safe chemical agents. More recently, a gene has been introduced into tomato that delays overripening and prolongs shelf life of the fruit.
Other traits of interest include those associated with grain quality. Genes to increase the content of amino acids such as lysine, methionine, and tryptophan in seed will increase nutritional value, thereby decreasing the need for amending grains with costly feed supplements.
All traits discussed here are associated with expression of single genes. But many important agronomic traits such as yield and lodging are not well understood and are controlled by many genes. Manipulating such polygenic traits by genetic engineering will require further research and the development of techniques for isolating, reconstructing, and transferring complex blocks of genes. Extensive and promising research is being conducted about additive disease resistance and stress tolerance, important polygenic traits. Plant genetic engineering is thus moving slowly but steadily from the laboratory bench into the field.
Matthew B. Wheeler, assistant professor of animal sciences; Stephen K. Farrand, professor of plant pathology and microbiology; and Jack M. Widholm, professor of plant physiology, Department of Agronomy
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